Biology subjects

Frederick, J.

Publications and source records attributed to Frederick, J..

14 recordsLinked to original sources

bioRxiv2024

Leveraging chromatin packing domains to target chemoevasion in vivo

Cancer cells exhibit a remarkable resilience to cytotoxic stress, often adapting through transcriptional changes linked to alterations in chromatin structure. In several types of cancer, these adaptations involve epigenetic modifications and restructuring of topologically associating domains (TADs). However, the underlying principles by which chromatin architecture facilitates such adaptability across different cancers remain poorly understood. To investigate the role of chromatin in this process, we developed a physics-based mechanistic model that connects chromatin organization to cell fate decisions, specifically survival following chemotherapy. Our model builds on the observation that chromatin forms packing domains, which influence transcriptional efficiency through macromolecular crowding. The model accurately predicts chemoevasion in vitro, suggesting that changes in packing domains affect the likelihood of survival. Consistent results across diverse cancer types indicate that the model captures fundamental principles of chromatin-mediated adaptation, independent of the specific cancer or chemotherapy mechanisms involved. Based on these insights, we hypothesized that compounds capable of modulating packing domains, termed Transcriptional Plasticity Regulators (TPRs), could prevent cellular adaptation to chemotherapy. Using live-cell chromatin imaging, we conducted a compound screen that identified several TPRs which synergistically enhanced chemotherapyinduced cell death. The most effective TPR significantly improved therapeutic outcomes in a patient-derived xenograft (PDX) model of ovarian cancer. These findings underscore the central role of chromatin in cellular adaptation to cytotoxic stress and present a novel framework for enhancing cancer therapies, with broad potential across multiple cancer types.

biophysics↗

bioRxiv2024

Three-color single-molecule localization microscopy in chromatin

Super-resolution microscopy has revolutionized our ability to visualize structures below the diffraction limit of conventional optical microscopy and is particularly useful for investigating complex biological targets like chromatin. Chromatin exhibits a hierarchical organization with structural compartments and domains at different length scales, from nanometers to micrometers. Single molecule localization microscopy (SMLM) methods, such as STORM, are essential for studying chromatin at the supra-nucleosome level due to their ability to target epigenetic marks that determine chromatin organization. Multi-label imaging of chromatin is necessary to unpack its structural complexity. However, these efforts are challenged by the high-density nuclear environment, which can affect antibody binding affinities, diffusivity and non-specific interactions. Optimizing buffer conditions, fluorophore stability, and antibody specificity is crucial for achieving effective antibody conjugates. Here, we demonstrate a sequential immunolabeling protocol that reliably enables three-label studies within the dense nuclear environment. This protocol couples multiplexed localization datasets with a robust analysis algorithm, which utilizes localizations from one target as seed points for distance, density and multi-label joint affinity measurements to explore complex organization of all three targets. Applying this multiplexed algorithm to analyze distance and joint density reveals that heterochromatin and euchromatin are not-distinct territories, but that localization of transcription and euchromatin couple with the periphery of heterochromatic clusters. This work is a crucial step in molecular imaging of the dense nuclear environment as multi-label capacity enables for investigation of complex multi-component systems like chromatin with enhanced accuracy.

bioengineering↗

bioRxiv2024

The repetitive genome of the Ixodes ricinus tick reveals transposable elements have driven genome evolution in ticks

Ticks are obligate blood-feeding parasites associated with a huge diversity of diseases globally. The hard tick Ixodes ricinus is the key vector of Lyme borreliosis and tick-borne encephalitis in Western Eurasia. Ixodes ticks have large and repetitive genomes that are not yet well characterized. Here we generate two high-quality I. ricinus genome assemblies, with haploid genome sizes of approximately 2.15 Gbp. We find transposable elements comprise at least 69% of the two I. ricinus genomes, amongst the highest proportions found in animals. The transposable elements in ticks are highly diverse and novel, so we constructed a repeat library for ticks using our I. ricinus genomes and the genome of I. scapularis, another major tick vector of Lyme borreliosis. To understand the impact of transposable elements on tick genomes we compared their accumulation in the two Ixodes sister species. We find transposable elements in these two species to be drivers of genome evolution in ticks. The I. ricinus genome assemblies and our tick repeat library will be valuable resources for biological insights into this important ectoparasite. Our findings highlight that further research into the impact of transposable elements on the genomes of blood-feeding parasites is required.

genomics↗

bioRxiv2024

Formamide denaturation of double-stranded DNA for fluorescence in situ hybridization (FISH) distorts nanoscale chromatin structure

As imaging techniques rapidly evolve to probe nanoscale genome organization at higher resolution, it is critical to consider how the reagents and procedures involved in sample preparation affect chromatin at the relevant length scales. Here, we investigate the effects of fluorescent labeling of DNA sequences within chromatin using the gold standard technique of three-dimensional fluorescence in situ hybridization (3D FISH). The chemical reagents involved in the 3D FISH protocol, specifically formamide, cause significant alterations to the sub-200 nm (sub-Mbp) chromatin structure. Alternatively, two labeling methods that do not rely on formamide denaturation, resolution after single-strand exonuclease resection (RASER)-FISH and clustered regularly interspaced short palindromic repeats (CRISPR)-Sirius, had minimal impact on the three-dimensional organization of chromatin. We present a polymer physics-based analysis of these protocols with guidelines for their interpretation when assessing chromatin structure using currently available techniques.

biophysics↗

bioRxiv2024

Microgroove substrates unveil topography-driven, dynamic 3D nuclear deformations

Navigating complex extracellular environments requires extensive deformation of cells and their nuclei. Nuclear deformations are intricately linked to nuclear structure and mechanical properties, and abnormalities in nuclear mechanics contribute to various diseases including laminopathies and cancer. Most in vitro systems used to study nuclear deformations are typically designed to generate strong whole-cell confinement relevant for specific cell types such as immune or cancer cells. Here, we use microgroove substrates as a model of anisotropic basement membrane topography and we report that adherent cells including endothelial cells and myoblasts exhibit significant 3D (in-plane and out-of-plane) nuclear deformations, with partial to complete penetration into the microgrooves. These deformations are dynamic with nuclei cyclically entering and exiting the microgrooves. AFM measurements show that these deformation cycles are accompanied by transient changes in nuclear mechanical properties. We also show that nuclear penetration into the grooves is principally driven by cell-substrate adhesion, without the need for cytoskeleton-associated forces. Finally, we demonstrate that myoblasts from patients with LMNA mutations exhibit abnormal nuclear deformations which can be rapidly identified and quantified using automated image analysis. We therefore propose the use of microgrooves as a novel simple, tunable, and high throughput system to study nuclear deformations in adherent cells, with the potential to serve as a functional diagnostic platform for pathological alterations in nuclear mechanics.

cell biology↗

bioRxiv2023

Tunable biomimetic materials elaborated by ice templating and self-assembly of collagen for tubular tissue engineering

Synthetic tubular grafts currently used in clinical context fail frequently, and the expectations that biomimetic materials could tackle these limitations are high. However, developing tubular materials presenting structural, compositional and functional properties close to those of native tissues remains an unmet challenge. Here we describe a combination of ice templating and topotactic fibrillogenesis of type I collagen, the main component of tissues extracellular matrix, yielding highly concentrated yet porous tubular collagen materials with controlled hierarchical architecture at multiple length scales, the hallmark of native tissues organization. By modulating the thermal conductivity of the cylindrical molds, we tune the macroscopic porosity defined by ice. Coupling the aforementioned porosity patterns with two different fibrillogenesis routes results in a new family of materials whose textural features and the supramolecular arrangement of type I collagen are achieved. The resulting materials present hierarchical elastic properties and are successfully colonized by human endothelial cells and alveolar epithelial cells on the luminal side, and by human mesenchymal stem cells on the external side. The results reported here demonstrate the relevance of the proposed straightforward protocol, likely to be adapted for larger graft sizes, to address ever-growing clinical needs such as peripheral arterial disease or tracheal and bronchial reconstructions.

bioengineering↗

bioRxiv2023

Multiple mechanisms contribute to isolation-by-environment in the redheaded pine sawfly, Neodiprion lecontei

Isolation by environment (IBE) is a population genomic pattern that arises when ecological barriers reduce gene flow between populations. Although current evidence suggests IBE is common in nature, few studies have evaluated the underlying mechanisms that generate IBE patterns. In this study, we evaluate five proposed mechanisms of IBE (natural selection against immigrants, sexual selection against immigrants, selection against hybrids, biased dispersal, environment-based phenological differences) that may give rise to host-associated differentiation within a sympatric population of the redheaded pine sawfly, Neodiprion lecontei, a species for which IBE has previously been detected. We first characterize the three pine species used by N. lecontei at the site, finding morphological and chemical differences among the hosts that could generate divergent selection on sawfly host-use traits. Next, using morphometrics and ddRAD sequencing, we detect modest phenotypic and genetic differentiation among sawflies originating from different pines that is consistent with recent, in situ divergence. Finally, via a series of laboratory assays - including assessments of larval performance on different hosts, adult mate and host preferences, hybrid fitness, and adult eclosion timing - we find evidence that multiple mechanisms contribute to IBE in N. lecontei. Overall, our results suggest IBE can emerge quickly, possibly due to multiple mechanisms acting in concert to reduce migration between different environments.

evolutionary biology↗

bioRxiv2023

Local compression of the root in a microfluidic device triggers a calcium signal

Throughout their life, plant root are submitted to mechanical stresses due to pressure exerted by the soil. So far, few studies addressed root cell deformation and calcium signaling elicited by soil compression. In this study, we designed a microchip inspired by pneumatic microvalve concept in order to deliver a lateral pressure to the root of a plant expressing the RGECO1-mTurquoise calcium reporter. Lateral pressure applied on the root induced a moderate elastic deformation of root cortical cells and elicited a multicomponent calcium signal at the onset of the pressure pulse, followed by a second one at the release of the pressure. This indicates that straining rather than stressing of tissues is relevant to trigger the calcium signal. The calcium elevation was restricted to the tissue under pressure and did not propagate. Additionally, the calcium signals exhibited a remarkable attenuation upon repetitive stimulations. Highlights- A microvalve concept mimicking lateral soil pressure was developed. - Non-damaging lateral compression of the root induces an elastic deformation of cortical cells. - A multicomponent calcium signal is elicited at the onset of a pressure pulse and upon release of the pressure. - Straining rather than stressing of tissues is relevant to trigger the calcium signal. - The calcium signal is localized at the tissue under pressure and does not propagate. - Calcium signals exhibit a remarkable attenuation upon repetitive stimulations.

plant biology↗

bioRxiv2022

Functional mRNA delivery to hematopoietic stem and progenitor cells in vivo.

Gene correction of hematopoietic stem cells (HSC) is a promising therapeutic approach for multiple disorders. Current methods, however, require HSC collection from patients, gene correction during ex vivo culture, and re-infusion of corrected HSC into patients conditioned with chemotherapeutic agents. These approaches are complex, and the conditioning creates toxicities. We show that a lipid nanoparticle (LNP) can deliver mRNA encoding a reporter or a gene editing protein to HSC, with one injection transfecting [~]25% of mouse HSC, and repeated doses resulting in higher editing efficiencies. We also demonstrate LNP-driven in vivo mRNA delivery to HSC in non-human primates and humanized mice. These results demonstrate a translatable approach to deliver mRNA encoding therapeutic proteins, or gene correcting tools, to HSC that do not require cell culture or toxic conditioning. One-Sentence SummaryLNP can deliver functional mRNA to mouse, non-human primate, and human HSC.

immunology↗

bioRxiv2022

Spatially resolved human kidney multi-omics single cell atlas highlights the key role of the fibrotic microenvironment in kidney disease progression

Kidneys possess one of the most intricate three-dimensional cellular structures in the body, yet the spatial and molecular principles of kidney health and disease remain inadequately understood. Here, we have generated high-quality datasets for 81 samples, including single cell (sc), single nuclear (sn), spot level (Visium) and single cell resolution (CosMx) spatial (sp)-RNA expression, and sn open chromatin, capturing cells from healthy, diabetic, and hypertensive diseased human kidneys. By combining the snRNA, snATAC and scRNA sequencing we identify cell types and map these cell types to their locations within the tissue. Unbiased deconvolution of the spatial data identifies 4 distinct spatial microenvironments: glomerular, immune, tubule and fibrotic. We describe the complex, heterogenous cellular and spatial organization of human microenvironments in health and disease. Further, we find that the fibrotic microenvironment spatial gene signature is not only able to molecularly classify human kidneys, but it also offers an improved prognosis prediction compared to traditional histopathological analysis. We provide a comprehensive spatially resolved molecular roadmap of the human kidney and the fibrotic process, demonstrating the clinical utility of spatial transcriptomics.

genetics↗

bioRxiv2021

Chromatin forms nanoscale three-dimensional packing domains with fractal-like scaling behavior in vitro

Chromatin organization over a wide range of length scales plays a critical role in the regulation of transcription and deciphering the interplay of these processes requires high-resolution, three-dimensional, quantitative imaging of chromatin structure in vitro. Herein, we introduce ChromSTEM, a method that utilizes high angle annular dark-field imaging and tomography in scanning transmission electron microscopy combined with DNA-specific staining for electron microscopy. We utilized ChromSTEM to quantify chromatin structure in cultured cells and the statistical packing behavior of the chromatin polymer. Using chromatin mass and density analysis, we observed that chromatin forms spatially well-defined higher-order domains which are around 100 nm in radius, with a radially decreasing mass-density from the center to the periphery. Although the morphological properties of the domains vary within the same cell line, they seem to exhibit greater heterogeneity across cell lines, underlying a potential role of statistical chromatin packing in regulating cell-type-specific gene expression.

genomics↗

bioRxiv2021

Spike-in normalization for single-cell RNA-seq reveals dynamic global transcriptional activity mediating anti-cancer drug response

The transcriptional plasticity of cancer cells promotes intercellular heterogeneity in response to anti-cancer drugs and facilitates the generation of subpopulation surviving cells. Characterizing single-cell transcriptional heterogeneity after drug treatments can provide mechanistic insights into drug efficacy. Here we used single-cell RNA-seq to examine transcriptomic profiles of cancer cells treated with paclitaxel, celecoxib, and the combination of the two drugs. By normalizing the expression of endogenous genes to spike-in molecules, we found that celluar mRNA abundance shows dynamic regulation after drug treatment. Using a random forest model, we identified gene signatures classifying single cells into three states: transcriptional repression, amplification, and control-like. Treatment with paclitaxel or celecoxib alone generally repressed gene transcription across single cells. Interestingly, the drug combination resulted in transcriptional amplification and hyperactivation of mitochondrial oxidative phosphorylation pathway linking to enhanced cell killing efficiency. Finally, we identified a regulatory module enriched with metabolism and inflammation-related genes activated in a subpopulation of paclitaxel-treated cells, the expression of which predicted paclitaxel efficacy across cancer cell lines and in vivo patient samples. Our study highlights the dynamic global transcriptional activity driving single-cell heterogeneity during drug response and emphasizes the importance of adding spike-in molecules to study gene expression regulation using single-cell RNA-seq.

genomics↗

bioRxiv2020

Nanoscale Chromatin Imaging and Analysis (nano-ChIA) platform bridges 4-D chromatin organization with molecular function

In eukaryotic cells, chromatin structure is linked to transcription processes through the regulation of genome organization. Extending across multiple length-scales - from the nucleosome to higher-order three-dimensional structures - chromatin is a dynamic system which evolves throughout the lifetime of a cell. However, no individual technique can fully elucidate the behavior of chromatin organization and its relation to molecular function at all length- and timescales at both a single-cell and a cell population level. Herein, we present a multi-technique nanoscale Chromatin Imaging and Analysis (nano-ChIA) platform that bridges electron tomography and optical superresolution imaging of chromatin conformation and transcriptional processes, with resolution down to the level of individual nucleosomes, with high-throughput, label-free analysis of chromatin packing and its dynamics in live cells. Utilizing nano-ChIA, we observed that chromatin is localized into spatially separable packing domains, with an average diameter of around 200 nm, sub-Mb genomic size, and an internal fractal structure. The chromatin packing behavior of these domains is directly influenced by active gene transcription. Furthermore, we demonstrated that the chromatin packing domain structure is correlated among progenitor cells and all their progeny, indicating that the organization of chromatin into fractal packing domains is heritable across cell division. Further studies employing the nano-ChIA platform have the potential to provide a more coherent picture of chromatin structure and its relation to molecular function.

genomics↗

bioRxiv2019

Quantifying Three-dimensional Chromatin Organization Utilizing Scanning Transmission Electron Microscopy: ChromSTEM

Chromatin organization over a wide range of length scales plays a critical role in the regulation of gene expression and deciphering these processes requires high-resolution, three-dimensional, quantitative imaging of chromatin structure in vitro. Herein we introduce ChromSTEM, a method which utilizes high angle annular dark field imaging and tomography in scanning transmission electron microscopy in combination with DNA-specific staining for electron microscopy. We utilized ChromSTEM to quantify chromatin structure in cultured cells and tissue biopsies through local DNA distribution and the scaling behavior of chromatin polymer. We observed that chromatin is densely packed with an average volume concentration of over 30% with heterochromatin having a two-fold higher density compared to euchromatin. Chromatin was arranged into spatially well-defined nanoscale packing domains with fractal internal structure and genomic size between 100 and 400 kb, comparable to that of topologically associated domains. The packing domains varied in DNA concentration and fractal dimension and had one of the distinct states of chromatin packing with differential ratio of DNA content to the chromatin volume concentration. Finally, we observed a significant intercellular heterogeneity of chromatin organization even within a genetically uniform cell population, which demonstrates the imperative for high-throughput characterization of chromatin structure at the single cell level.

genomics↗